1. Field of the Invention
The present invention relates generally to synthetic substrates, more particularly to synthetic substrates useful in qualitative and quantitative methods for determining proteolytic enzymes.
2. Prior Art
In recent years the study of proteinases has been greatly advanced by the recognition that construction of synthetic substrates to simulate segments of naturally occurring substrates can lead to remarkably comparable specificity of enzyme activity. Although proteins are the natural substrates for proteinases, they are often unsuitable for detecting enzyme activity or presence because they offer multiple sites for enzyme cleavage. On the other hand, synthetic substrates have well defined chemical structures and the kinetics of hydrolysis are simplified when there is only a single point of cleavage. Thus, enzyme specificity may be determined by the number and arrangement of amino acids in the peptide moiety of the synthetic substrate, and the activity of the enzyme may be measured by the amount of a detector moiety liberated upon hydrolysis of the synthetic substrate.
Particular synthetic substrates have been found useful in determinations of specific enzymes in biological fluids such as blood, serum, urine, and the like. For example, Svendsen et al reported use of synthetic substrates in the assay of thrombin and thrombin-like enzymes. "Synthetic chromogenic substrates for determination of trypsin, thrombin and thrombin-like enzymes", Thromb. Res., 1:267-278 (1972). More recently, Kirchof et al have reported improvements in measuring the prothrombin concentration in plasma, using synthetic substrates, for clinical applications such as aiding in the control of anticoagulant therapy. "Control of an anticoagulant therapy with a chromogenic substrate", Haemostasis, 8: 1-7 (1979).
To date there are six, generally accepted detector groups for such synthetic substrates, each of which has unique properties and which may be better suited to assay a particular proteinase than the others. These six common detector groups are: para-nitroaniline; 2-naphthylamine; 4-methoxy-2-naphthylamine; 5-aminoisophthalic acid; 7-amino-4-methylcoumarin; and, 7-amino-4-trifluoromethylcoumarin. The latter five detector groups may be detected by either fluorometric or colorimetric methods. Because the chromophore, para-nitroaniline, is yellow, enzyme assays employing this chromosphore are normally colorimetric.
These known substrates for quantification of enzymes in solution, i.e., blood, serum, urine or the like, have not been satisfactory for enzyme localization within monodispersed cells. One recent report suggests that quantification of enzymes in single cells appears promising by fluorescent staining, and discloses the use of coupling 4-methoxy-2-naphthylamine to 5-nitrosalicylaldehyde. R. E. Smith and P. N. Dean, Journal of Histochemistry and Cytochemistry, Vol. 27, No. 11, pp. 1499-1504 (1979).
The identification and quantification of proteinases in single cells holds great potential for applications such as diagnosing disease states, but to date has been hindered by the lack of suitable detector groups. This is believed due at least in part to the solubility of the known detector groups which, when liberated from the synthetic substrates, have tended to permeate the cell membrane rather than remain localized within the cell.